This study combines a cellular and molecular analysis of the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) to determine the osmoregulatory role of this protein in different tissues during the ontogeny of the sea bass. We have characterized the complete sequence of the NKCC1 isoform isolated from the sea bass gills and have identified, by immunofluorescence, NKCC1, and other isoforms, within the epithelium of the major osmoregulatory organs. Different (absorptive and secretory) functions have been attributed to this protein according to the tissue and salinity. The effects of short- (1-4 days), medium- (7-21 days) and long (6 months)-term freshwater (FW) adaptations were investigated, in comparison with seawater (SW)-maintained sea bass. In adult sea bass after long-term adaptation to FW and SW, the gills had the highest expression of NKCC mRNA compared with the median/posterior kidney and to the posterior intestine. Expression of NKCC mRNA in the kidney was 95% (SW) and 63% (FW) lower, and in the intestine 98% (SW) and 77% (FW) lower. Compared to SW-maintained sea bass, long-term FW adaptation induced a significant 5.6-fold decrease in the branchial NKCC gene expression whereas the intestinal and renal expressions did not vary significantly. The cells of the intestine and collecting ducts as well as a part of the epithelium lining the urinary bladder expressed NKCC apically. Within the gill chloride cells, NKCC was found basolaterally in SW-acclimated fish; some apically stained cells were detected after 7 days of FW exposure and their relative number increased progressively following FW acclimation. The appearance of FW-type chloride cells induces a functional shift of the gills from a secretory to an absorptive epithelium, which was only completed after long-term exposure to FW. Short- and medium-term exposure to FW induced a progressive decrease in total NKCC content and an increase in functionally different branchial chloride cells. During development, the cotransporter was already expressed in tegumentary ionocytes and along the digestive tract of late embryos. NKCC was recorded in the branchial chamber and along the renal collecting ducts in prelarvae and also in the dorsal part of the urinary bladder in larvae. The expression of NKCC along the osmoregulatory epithelial cells and the presence of Na(+)/K(+)-ATPase within these cells contribute to the increase of the osmoregulatory capacity during sea bass ontogeny.